PROJECT SUMMARY This career development proposal describes a 5-year mentored research project which will enhance our understanding of Klebsiella pneumoniae (Kp) pathogenesis in the lung and subsequent host immune response. The project will build upon the candidate's background in bacteriology, pathogenesis of Gram- negative organisms, and infectious diseases. A combination of didactic and practical training, detailed in the comprehensive career development plan, will provide the candidate with enhanced training in lung immunobiology, adaptive immunity, and bacterial signaling, and technical training in methodologies such as flow cytometry and ChIP-Seq. Successful completion of the proposed aims will enable the candidate to gain the skills required to secure independent funding and transition as an independent physician-scientist. The candidate will benefit from the superb mentorship of David A. Hunstad, MD, and John P. Atkinson, MD. In established and newly created murine models, Dr. Hunstad has extensively dissected host-pathogen interactions and host immune responses in Gram-negative bacterial infections, while Dr. Atkinson is a world- renowned immunologist with extensive experience in training physician-scientists. The candidate is supported by a world-class advisory committee, each member chosen for specific expertise relevant to the research proposal and career development plan. The work will take place in the tremendous Washington University environment with its abundant scientific resources and rich history in the training of physician-scientists. Kp infections, including pneumonia, urinary tract infection, and bloodstream infection, are sharply on the rise in hospitalized patients; CDC has recently declared that infections with Kp and other carbapenem- resistant Enterobacteriaceae (CRE) demand a threat level of urgent. Two important Kp virulence factors, capsule and type 1 pili, are known to be important in distinct host niches, specifically the lung and urinary tract, respectively. The candidate will test the hypothesis that these virulence factors are coordinately and inversely regulated, and will identify the mechanistic link underlying this regulation. The candidate will determine how type 1 pili, their phase switch fimS, regulator FimK, and the bacterial second messenger cyclic-di-GMP affect capsule production in Kp and downstream virulence in a murine model of Kp pneumonia. In addition, despite the prevalence and clinical importance of Kp, little is known about natural protective immunity against Kp. Using a new model strain of Kp, the candidate's preliminary data indicate that Kp infection of the lungs in mice is protective from subsequent infection. Lymphocytic infiltrates resembling inducible bronchus-associated lymphoid tissue are observed in the lungs of these protected hosts. Thus, the proposed research will also characterize the adaptive immune response to Kp pneumonia and the role of capsule in eliciting protection.These studies may identify bacterial targets amenable to anti-virulence therapeutics, while also laying the foundation for potential vaccine development to prevent serious Kp infection.